Quaternary ammonium compounds, (C16-18 and C18-unsatd. alkyl)trimethyl, chlorides

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 17 July, 2009 to 21 August, 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

yes

Remarks:

minor deviations: (a) ammonium chloride was not added to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound), and b) river water was used as inoculum.

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

other: River water without particles

Details on inoculum:

River water was sampled from the Rhine near Heveadorp, The Netherlands 1707-2009). The river water was aerated for 7 days before use to reduce the endogenous respiration (van Ginkel and Stroo, 1992). River water without particles was used as inoculum. The particles were removed by sedimentation.

As per the guidelines, activated sludge, effluent from biological wastewater treatment plant, river water, soil etc. are suggested as inocula. Possible characterization of the inoculum for instance by determining the most probable number (MPN) is not obligatory. This is because the ecosystems from which the inoculum are derived change continuously over time; hence not making it possible to have identical or constant amounts of inoculum at every sampling.
However, the use of the small inoculum can be justified based on the fact that the test substance fulfills the validity criteria ≤ 1.5 mg/L oxygen consumption in the control group at Day 28 in the Closed Bottle test. This criteria fulfilment indicates low endogenous respiration, which is due to presence of small amount of inoculum. Further, the river water was used undiluted and was not pre-exposed to the test substance after sampling, which is another critical pre-requisite for ready biodegradation tests with regard to the inoculum.
Therefore, based on the above arguments, the absence of specification of inoculum concentration will not pose as a hindrance in evaluating the ready biodegradation results of the registered substance.

Duration of test (contact time):

ca. 28 d

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Nutrients and stock solutions: The river water used in the closed bottle test was spiked per liter of water with 8.5 mg KH2P04, 21.75 mg K2HP04, 33.3 mg Na2HP04·2H20, 22.5 mg MgS04·7H20, 27.5 mg CaCI2, 0.25 mg FeCl3·6H20. Ammonium chloride was not added to prevent nitrification. Sodium acetate and the test substance were added to the bottles using stock solutions of 1.0 g/L.
- Deionized water: Deionized water containing no more than 0.01 mg/L Cu (Sterlab certified; nonGLPanalysis) was prepared in a water purification system.
- Test temperature: 22(21.8) to 24°C.
- pH: 7.9


TEST SYSTEM
- Test bottles: The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.
- Number of culture flasks/concentration: 10 bottles containing only river water (inoculum and medium), 10 bottles containing river water and silica gel (2 g/bottle), 10 bottles containing river water, silica gel and test substance (2 g/L), and 6 bottles containing sodium acetate (6.7 mg/L) and river water. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles.

SAMPLING
- Sampling frequency: The zero time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at Day 7, 14, 21, and 28.

ANALYSES
The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode (WTW Trioxmatic EO 200) and meter (WTW OXI 530) (Retsch,
Ochten, The Netherlands). The pH was measured using a Knick 765 calimatic pH meter (Elektronische Messgerate GmbH, Berlin, Germany). The temperature was
measured and recorded with a sensor connected to a data logger.

Reference substance:

acetic acid, sodium salt

Remarks:

at 6.7 mg/L

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNH3)

Value:

71

Sampling time:

28 d

Remarks on result:

other: readily biodegradable

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

69

Sampling time:

28 d

Remarks on result:

other: readily biodegradable

Details on results:

THEORETICAL OXYGEN DEMAND (ThOD):
The calculated ThOD of the test substance was 2.9 mg/mg. The ThOD of the test substance composed of the active substance, 2-propanol and water was 2.3 mg/mg. The ThOD of sodium acetate was 0.8 mg/mg.

TOXICITY:
Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test substance in the Closed Bottle test was not determined because
possible toxicity of the test substance to microorganisms degrading acetate is not relevant. Inhibition of the endogenous respiration of the inoculum by the test substance tested in the presence of silica gel was not detected. Therefore, no inhibition of the biodegradation due to the "high" initial concentration of the test substance is expected.

VALIDITY OF THE TEST:
The validity of the test was demonstrated by an endogenous respiration of 1.0 mg/L at Day 28. Furthermore, the differences of the replicate values at Day 28 were less than 20%. The biodegradation percentage of the reference compound, sodium acetate, at Day 14 was 76. Finally, the validity of the test was shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.
Please refer to the tables for details under 'attached background materials'

BIODEGRADABILITY:
The test substance only fulfilled one criterion for ready biodegradable compounds i.e. a biodegradation percentage in excess of 60 within 28 d. The pass level of 60% was not reached within 10 d upon achieving 10% biodegradation. The reasons for failing the 10-d window are the composition of the test substance (TMAC and 2-propanol) and TMAC being a chemical consisting of a hydrophilic group linked to a hydrophobic moiety. Biodegradation of both moieties of surfactants requires the concerted action of at least two microorganisms, as a single organism usually lacks the full complement of enzymatic capabilities (van Ginkel, 1996). In ready biodegradability tests, the two moieties of this fatty amine derivative are therefore degraded sequentially. The time window criterion was developed on the assumption that a compound is degraded according to the "standard" growth curve in ready biodegradability tests.

The degradation curve of the test substance is the sum of growth curves of 2-propanol and the two moieties. The biodegradability of 2-propanol and the two moieties in the closed bottle test may be fully in line with the timeday window criterion when judged as separate chemicals. The time-window should therefore be ignored as a pass fail criterion for TMAC contained in the test substance. The test substance was therefore classified as readilybiodegradable only based on the biodegradation percentage of 71% at Day 28.

Results with reference substance:

The biodegradation percentage of the reference compound, sodium acetate, at Day 14 was 76.

For result tables and figure, kindly refer to the attached background material section of the IUCLID.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Under the study conditions, the test substance is considered to be readily biodegradable.

Executive summary:

A study was conducted to determine the ready biodegradability of the test substance, TMAC T (49% active in isopropanol and water), in water according to OECD Guideline 301D (closed bottle test), in compliance with GLP. The test substance at 2 mg/L was incubated with inoculums from river water and O2 consumption was followed over a period of 28 days. The test was performed using 10 bottles containing only river water (inoculum and medium), 10 bottles containing river water and silica gel (2 g/bottle), 10 bottles containing river water, silica gel and test substance, and 6 bottles containing sodium acetate and river water. The concentrations of the test substance and sodium acetate in the bottles were 2.0 and 6. 7 mg/L, respectively. Each of the prepared solutions was dispensed into the respective group of 0.3 L BOD bottles so that all bottles were completely filled without air bubbles. The zero-time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at Day 7, 14, 21, and 28. The test was found to be valid as shown by an endogenous respiration of 1.0 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 76% of its theoretical oxygen demand after 14 day. Finally, the most important criterion was met with the oxygen concentrations being > 0.5 mg/L in all bottles during the test period. Biodegradability of the test substance was determined to be 71% and 69% by the end of 28 days using ThODNH3 and ThODNO3 equations respectively. Therefore, the substance can be considered readily biodegradable in water. Furthermore, the test substance did not cause a reduction in the endogenous respiration in presence of silica gel, hence was considered to be non-inhibitory to the inoculum. Under the conditions of the study, the test substance is considered to be readily biodegradable (van Ginkel CG, 2010).

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Remarks:

Preliminary screening

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Preliminary test

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Version / remarks:

(preliminary test)

Deviations:

not specified

GLP compliance:

no

Remarks:

Preliminary non-GLP study; main study has been planned to be conducted under GLP conditions.

Oxygen conditions:

aerobic

Inoculum or test system:

other: activated sludge, domestic, non-adapted and river water

Details on inoculum:

(a) Activated sludge wasobtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant treats predominantly domestic wastewater. The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 0.40 g Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted to 2.0 mg DW/in the biological oxygen demand (BOD) bottles (van Ginkel and Stroo, 1992).
(b) River water was sampled from the Rhine near Heveadorp, The Netherlands. The river water was aerated for 7 days to reduce the endogenous respiration. River water without particles was used as inoculum. The particles were removed by sedimentation after 1 day while moderately aerating. The river water spiked with mineral salts of the nutrient medium was used undiluted.
The Colony forming units (CFU) of the preconditioned river water inoculum and diluted preconditioned activated sludge inoculum was determined by a colony count method based on the ISO 6222 (1999) guideline. The preconditioned and diluted inoculum as used in the closed bottles was diluted 10x and 100x in a sterile peptone solution (1 g/L). Subsequently 1 ml of the peptone dilutions was transferred on a sterile petri dish and yeast extract agar was added. The yeast extract agar contained per liter of water 6 g tryptone, 3 g yeast extract and 15 g agar. Yeast extract agar plates were incubated for 68 hours at a temperature ranging from 22.7 – 22.9 °C. Only CFU counts between 30 and 300 were regarded as accurate and accepted for calculation of the CFU content. The inoculum concentration in the bottles determined by colony count was 7E+5 CFU/L and 6E+5 CFU/L for the river water and activated sludge inoculum, respectively.

Duration of test (contact time):

ca. 42 d

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Test procedures of Closed Bottle test
The Closed Bottle test was performed according to Test Guidelines (OECD 1992). The nutrient medium of the Closed Bottle test contained per liter of deionized water: 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.4 mg Na2HPO4·2H2O, 22.5 mg MgSO4·7H2O, 27.5 mg CaCl2, and 0.25 mg FeCl3·6H2O, Ammonium chloride was omitted from the medium to prevent nitrification. To perform these “negative control” tests without deviations from the guideline 0.5 mg/L ammonium chloride was included in the sorbent free tests. Test substance and humic acid were dosed using an aqueous stock solution of 1 g/L in water. Isopropanol was dosed from a 0.1 g/L stock solution in demiwater. The tests were performed in 0.3 L BOD bottles with glass stoppers. In the tests without sorbent use was made of 3 bottles with the test substance and the respective inoculum and 3 control bottles only containing the respective inoculum and 36 µg/L isopropanol (to correct for the small amount of isopropanol still present in the test substance). In the sorbent modified tests use was made of 3 bottles containing the test substance, the respective inoculum and silica gel or humic acid, and 3 control bottles containing only respective inoculum, 36 µg/L isopropanol (to correct for the small amount of isopropanol still present in the test substance), and silica gel or humic acid. The tests without sorbent were used to demonstrate the toxic effects of the test substance to the inoculum in the Closed Bottle test and to illustrate the positive detoxifying effects of the sorbents. Silicagel and humic acid concentrations in the bottles (test and control) were 1 and 2 g /bottle and 1 and 2 mg acid/L, respectively. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The bottles were closed and incubated in the dark at temperatures ranging from 22 to 24°C. The biodegradation was measured by following the course of the oxygen decrease in the bottles using a special funnel and an oxygen electrode. This funnel fitted exactly in the BOD bottle, when the oxygen electrode was inserted in the BOD bottle the funnel collected the dissipated medium. Upon the removal of the oxygen electrode the collected medium flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo 1992).

Analyses
The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter (WTW). The pH was measured using a EUTECH instruments pH meter. The temperature was measured and recorded with a thermo couple connected to a data logger. The dry weight of the inoculum was determined by filtrating 50 mL of the activated sludge over a pre-weighed 12 um cellulose nitrate filter. This filter was dried for 1.5 hours at 104°C and weighed after cooling. The dry weight was calculated by subtracting the weighed filters and by dividing this difference by the filtered volume.

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

44

Sampling time:

28 d

Remarks on result:

other: activated sludge as inoculum and without sorbent

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

61

Sampling time:

28 d

Remarks on result:

other: 65% after 42 d; (using activated sludge as inoculum and 2 g silica gel / bottle for detoxification)

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

65

Sampling time:

28 d

Remarks on result:

other: 67% after 42 d; (using activated sludge as inoculum and 1 g silica gel / bottle for detoxification)

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

60

Sampling time:

28 d

Remarks on result:

other: 61% after 42 d; (using activated sludge as inoculum and 2 mg/L humic acid / bottle for detoxification)

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

56

Sampling time:

28 d

Remarks on result:

other: 60% after 42 d; (using activated sludge as inoculum and 1 mg/L humic acid/ bottle for detoxification)

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

35

Sampling time:

28 d

Remarks on result:

other: river water as inoculum and without sorbent

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

47

Sampling time:

28 d

Remarks on result:

other: 51% after 42 d; (using river water as inoculum and 2 g silica gel / bottle for detoxification)

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

50

Sampling time:

28 d

Remarks on result:

other: 56% after 42 d; (using river water as inoculum and 1 g silica gel / bottle for detoxification)

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

54

Sampling time:

28 d

Remarks on result:

other: 67% after 42 d; (using river water as inoculum and 2 mg/L humic acid / bottle for detoxification)

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

53

Sampling time:

28 d

Remarks on result:

other: 67% after 42 d; (using river water as inoculum and 1 mg/L humic acid / bottle for detoxification)

Results

Test conditions

The validity of the test is demonstrated by oxygen concentrations >0.5 mg/L in all bottles during the test period and by differences of less than 20% for the triplicate values at day 28. Furthermore, the endogenous respiration at day 28 was <1.5 mg/L in the tests were ammonium chloride was omitted from the medium. In the tests with ammonium chloride in the medium (the “negative control” test) the endogenous respiration was 1.5 and 1.6 mg/L for the activated sludge and river water inoculum, respectively. The pH of the media was 7.4 (activated sludge) and 8.2 (river water) at the start of the test. The pH was 7.2±0.1 (activated sludge) and 8.0±0.1 (river water) at day 42. Temperatures ranged from22 to 24°C. The inhibition of biodegradation by the test substances is usually detected prior to the onset of the biodegradation through suppression of the endogenous oxygen consumption. Hampering of the biodegradation by inhibition of the endogenous respiration of the inoculum was clearly detected until day 7-14 in the sorbent free ready biodegradation tests. Silica gel and humic acid were added as sorbent for detoxification of the test substance.The humic acid sorbent still showed an inhibition of the endogenous respiration (negative biodegradation percentages) at day 7. Detoxification was most successful by the silica gel sorbents and no inhibition of the biodegradation due to the “high” initial test substance concentration is expected in the presence of silica gel (1 and 2 g/bottle).

The Closed Bottle test results

The ThOD_NH3 and ThOD_NO3 of the active ingredient (active with average chain length) used to calculate the biodegradation percentages was 2.86 g/g and 3.06 g/g, respectively. In OECD 301 tests growth-linked biodegradation takes place. This means that carbon and nitrogen is built into microorganisms (new biomass). Calculating the biodegradation by using the ThOD_NO3 assumes that all the organic nitrogen present in a test substance is transiently degraded to ammonium nitrogen and subsequently oxidized to nitrate. The C:N ratio of test substance (solvent free) is ~20:1. In the sorbent modified tests the organic nitrogen of the test substance is the only nitrogen present for growth due to the omission of ammonium nitrogen from the nutrient medium. With a C:N ratio of about 7:1 for growth of biomass (not exact) this would mean that most likely all the organic nitrogen present in the test substance will be built into new biomass. No oxidation of test substance nitrogen to nitrate is therefore expected in the CBT. The use of ThOD_NH3 can however only be justified if the low nitrate/nitrite concentrations in the CBT can be demonstrated by analysis. However, analysis is probably not accurate enough to demonstrate this small possible increase in nitrate/nitrite concentrations (maximum theoretical amount of NO₃formed from organic nitrogen present in the test substance is 0.4 mg/L and the “background” NO₃ concentration in CBT blank with activated sludge and river water is ~0.7 mg/L and ~10 mg/L, respectively). The biodegradation of test substance (solvent free) is for this reason calculated with the “worst-case approach” assuming a complete oxidation of the organic nitrogen to nitrate (using the ThOD_NO3). The biodegradation percentages at day 28 using activated sludge as inoculum were slightly higher compared to results achieved with river water. More than 60% biodegradation was still achieved within 28 days using activated sludge as inoculum. Test substance (solvent free) should therefore be classified as readily biodegradable. For the final GLP test it is recommended to use activated sludge as inoculum and 1 g silica gel /bottle for detoxification of the test substance.

Table I Percentages biodegradation of test substance (solvent free) in Closed Bottle tests inoculated with activated sludge and river water. Biodegradation percentages were calculated using the ThOD_NH3.

Inoculum	Sorbent	Biodegradation percentage at day
		7	14	21	28	42
Activated sludge	No sorbent*	-6	17	42	47	-
	2 g silica gel /bottle	10	42	52	65	69
	1 g silica gel / bottle	13	49	58	69	71
	2 mg/L humic acid	-2	35	52	64	65
	1 mg/L humic acid	-3	34	52	60	64
River water	No sorbent*	-10	-4	21	37	-
	2 g silica gel /bottle	2	37	47	50	54
	1 g silica gel / bottle	4	41	48	53	60
	2 mg/L humic acid	-6	36	53	57	72
	1 mg/L humic acid	-4	35	53	56	71

* NH₄Cl (0.5 mg/L) was included in the nutrient medium as prescribed in the OECD 301D guideline.

 

Table II Percentages biodegradation of test substance (solvent free) in Closed Bottle tests inoculated with activated sludge and river water. Biodegradation percentages were calculated using the ThOD_NO3.

Inoculum	Sorbent	Biodegradation percentage at day
		7	14	21	28	42
Activated sludge	No sorbent*	-5	16	39	44	-
	2 g silica gel /bottle	9	40	49	61	65
	1 g silica gel / bottle	13	46	54	65	67
	2 mg/L humic acid	-2	33	48	60	61
	1 mg/L humic acid	-3	32	49	56	60
River water	No sorbent*	-9	-4	20	35	-
	2 g silica gel /bottle	2	35	44	47	51
	1 g silica gel / bottle	3	39	45	50	56
	2 mg/L humic acid	-5	34	50	54	67
	1 mg/L humic acid	-3	33	50	53	67

* NH₄Cl (0.5 mg/L) was included in the nutrient medium as prescribed in the OECD 301D guideline.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Under the study conditions, the test substance was determined to be readily biodegradable.

Executive summary:

A preliminary non-GLP study was conducted to determine the best test conditions for conducting the closed bottle ready biodegradation study with the test substance, TMAC T (96% active), according to the OECD Guideline 301D. Due to the well-known toxicity of the quaternary substances, the test substance was evaluated using detoxification methods through the addition of the sorbents silica gel and humic acid at two different concentrations and two different inocula (activated sludge, river water).In addition, a sorbent free test group without any deviations from the guideline was included as a ‘negative control’ and to demonstrate the toxicity of the test substance and to demonstrate the positive detoxifying effects of the sorbents. Ammonium chloride was omitted from the medium to prevent nitrification for all groups except the sorbent free group. The inoculum concentration in the bottles determined by colony count was 7E+5 CFU/L and 6E+5 CFU/L for the river water and activated sludge inoculum, respectively.

The tests were performed in triplicates using 0.3 L BOD bottles with glass stoppers. In the tests ‘without sorbent’ use was made of 3 bottles with the test substance (at 2 mg/L) and the respective inoculum and 3 control bottles only containing the respective inoculum and 36 μg/L isopropanol (to correct for the small amount of isopropanol still present in the test substance). In the ‘sorbent modified’ tests use was made of 3 bottles containing the test substance (at 2 mg/L), the respective inoculum and silica gel or humic acid, and 3 control bottles containing only respective inoculum, 36 μg/L isopropanol, and silica gel or humic acid. Silicagel and humic acid concentrations in the bottles (test and control) were 1 and 2 g /bottle and 1 and 2 mg acid/L, respectively. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The bottles were closed and incubated in the dark at temperatures ranging from 22 to 24°C. The biodegradation was measured by following the course of the oxygen decrease in the bottles using a special funnel and an oxygen electrode.

The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter (WTW). The theoretical oxygen demand (ThOD) of test substance was calculated from its molecular formula and molecular weight. The BOD (mg/mg) of the test substance was calculated by dividing the oxygen consumption by the concentration of the test substance in the closed bottle. The ThODNH3 and ThODNO3 of the active ingredient (active with average chain length) used to calculate the biodegradation percentages was 2.86 g/g and 3.06 g/g, respectively. The biodegradation percentages at Day 28 using activated sludge as inoculum were slightly higher compared to results achieved with river water. Using the conservative ThODNO3 to calculate the biodegradation of test substance still >60% biodegradation was achieved within 28 days using activated sludge as inoculum and 1 g silica gel / bottle for detoxification.

The validity of the test is demonstrated by oxygen concentrations >0.5 mg/L in all bottles during the test period. The pH of the media was 7.4 and 7.2±0.1 (activated sludge) and 8.2 and 8.0±0.1 (river water) at the start and end of Day 42 of the test respectively. Temperatures ranged from 22 to 24°C. The inhibition of biodegradation by the test substances is usually detected prior to the onset of the biodegradation through suppression of the endogenous oxygen consumption and this was clearly detected until day 7-14 in the sorbent free ready biodegradation tests. The humic acid sorbent still showed an inhibition of the endogenous respiration (negative biodegradation percentages) at Day 7. Detoxification was most successful by the silica gel sorbents and no inhibition of the biodegradation due to the “high” initial test substance concentration is expected in the presence of silica gel (1 and 2 g/bottle).

Under the study conditions, the test substance was determined to be readily biodegradable and the use activated sludge as inoculum and 1 g silica gel /bottle for detoxification of the test substance was considered further for the main study (Geerts, 2020).

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 14 Feb 2020 to 16 Mar 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

yes

Remarks:

Ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound).

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

other: activated sludge, domestic, non-adapted

Details on inoculum:

Secondary activated sludge (10-02-2020) was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant is an activated sludge treatment plant treating predominantly domestic wastewater. The dry weight of the inoculum was determined by filtrating 50 mL of the activated sludge over a preweighed 12 µm cellulose nitrate filter. This filter was dried for 1.5 hour at 103.9 °C and weighed after cooling. Dry weight was calculated by subtracting the weight of the filters and dividing the difference by the filtered volume. The measured dry weight of the inoculum was 3.2 g/L.

The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end the inoculum was diluted in aerated Closed Bottle test medium to 0.4 g Dry weight (DW)/L of activated sludge and aerated for one week. The preconditioned inoculum was diluted further to a dry weight concentration of 2 mg/L in the BOD bottles (van Ginkel and Stroo, 1992). The Colony forming units (CFU) of the preconditioned and diluted inoculum was determined by a colony count method based on the ISO 6222 (1999) guideline.

The preconditioned and diluted inoculum as used in the closed bottles (2 mg/L dry weight) was diluted 10x and 100x in a sterile peptone solution (1 g/L). Subsequently 1 ml of the peptone dilutions was transferred on a sterile petri dish and yeast extract agar was added. The yeast extract agar contained per liter of water 6 g tryptone, 3 g yeast extract and 15 g agar. Yeast extract agar plates were incubated for 68 hours at a temperature ranging from 22.7 – 22.9 °C. Only CFU counts between 30 and 300 were regarded as accurate and accepted for calculation of the CFU content. The inoculum concentration in the BOD bottles determined by colony count was 1E+6 CFU/L.

Duration of test (contact time):

ca. 28 d

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Reference substances and chemicals
The silica gel Davisil grade 636, pore size 60A, 35-60 mesh particle size (MKCH4201) was purchased from Sigma-Aldrich. Isopropanol analytical grade (lot 1913436) was obtained from Fisher Chemicals. All other chemicals used were of reagent grade quality.

Deionized water
Deionized water containing <1.0 mg/L of organic carbon was prepared in a water purification system.

Test bottles
The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.

Nutrients and stocks
Deionized water used in the Closed Bottle test contained per liter of water 8.51 mg KH2PO4, 21.75 mg K2HPO4, 33.42 mg Na2HPO4·2H2O, 22.50 mg MgSO4·7H2O, 27.51 mg CaCl2, 0.25 mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrification. The test substance, sodium acetate and isopropanol were added to the bottles using aqueous stock solution of 1 g/L, 1 g/L and 0.1 g/L, respectively. Silica gel was added as sorbent in the test bottles for detoxification of the test substance at a concentration of 1 g silica gel / bottle. Next the bottles were filled with nutrient medium with inoculum and closed.

Test procedures
The Closed Bottle test (OECD TG 301D) was performed according to the study plan. The study plan was developed from ISO Test Guidelines (1994). Use was made of 10 bottles containing only inoculum, 10 bottles containing inoculum, silica gel and isopropanol, 10 bottles containing inoculum and silica gel with test substance, 6 bottles containing inoculum and sodium acetate. The concentrations of the test substance, and sodium acetate in the bottles were 2.0 mg/L and 6.7 mg/L, respectively. The concentration isopropanol added to the control bottles with silica gel was 35 µg/L which is comparable to the isopropanol content present in the test bottles. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The zero-time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28.

Analyses
The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter (WTW). The pH was measured using an Eutech pH meter. The temperature was measured and recorded with a sensor connected to a data logger.

Reference substance:

benzoic acid, sodium salt

Remarks:

Purity: 99.9%, Batch/lot number: BCBP8197V, Appearance: white powder

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNO3)

Value:

63

Sampling time:

28 d

Remarks on result:

other: readily biodegradable

Parameter:

% degradation (O2 consumption)

Remarks:

(based on ThODNH3)

Value:

67

Sampling time:

28 d

Remarks on result:

other: readily biodegradable

Results

Theoretical oxygen demand (ThOD)

The ThOD_NH3 and ThOD_NO3 of the test substance used to calculate the biodegradation percentages is 2.86 and 3.06 g oxygen/g active ingredient, respectively. These ThODs were calculated with an average molecular formula of the active ingredient (of the test substance) using an average alkyl chain length. The average alkyl chain length is calculated from the alkyl chain distribution. The ThOD of sodium acetate is 0.78 g oxygen/g sodium acetate.

 

Toxicity

Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test substance in the Closed Bottle test was not determined because possible toxicity of the test substances to microorganisms degrading acetate is not relevant. Inhibition of the endogenous respiration of the inoculum by the test substance at day 7 was not detected. Therefore, no inhibition of the biodegradation due to the "high" initial test substance concentration is expected.

 

Test conditions

The pH of the media for the control, control with silica gel, reference and test substance was 7.3 at the start of the test. The pH of the medium in the reference bottles measured at day 14 was 7.2. The pH of the medium at day 28 was 7.0 for the test and 7.1 for the control and control with silica gel. The temperature ranged from 22.7 to 22.9 °C which is within the prescribed temperature range of 22 to 24°C.

 

Validity of the test

The validity of the test is demonstrated by an endogenous respiration of 1.10 mg/L at day 28. Furthermore, the differences of the replicate values at day 28 were less than 20%. The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 75%. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

 

Biodegradability

Solvent free test substance was biodegraded by 67% (based on ThOD_NH3) at day 28 in the Closed Bottle test. Assuming complete nitrification, and calculating the biodegradation based on the ThOD_NO3 the test substance was biodegraded by 63% in the Closed Bottle test at day 28. Solvent free test substance is classified as readily biodegradable based on the >60% biodegradation reached at day 28.

Table I Dissolved oxygen concentrations (mg/L) in the closed bottles

Time (days)	Oxygen concentration (mg/L)
	Oc	Oa	Ocs	Ot
0	8.9	8.9	8.9	8.9
	8.9	8.9	8.9	8.9
Mean (M)	8.90	8.90	8.90	8.90
7	8.5	4.5	8.3	8.0
	8.5	4.6	8.4	8.4
Mean (M)	8.50	4.55	8.35	8.20
14	8.0	4.2	8.0	6.5
	8.1	4.1	8.0	6.4
Mean (M)	8.05	4.15	8.00	6.45
21	8.0		8.0	5.2
	8.0		8.0	5.1
Mean (M)	8.00		8.00	5.15
28	7.8		7.8	4.2
	7.8		7.8	4.0
Mean (M)	7.80		7.80	4.10

O_c Mineral nutrient solution with only inoculum.

O_cs Mineral nutrient solution with inoculum, silica gel and isopropanol

O_t Mineral nutrient solution with inoculum, test substance (2.0 mg/L test substance = 1.92 mg/L active ingredient) and silica gel

O_a Mineral nutrient solution with inoculum and sodium acetate (6.7 mg/L).

Table II Oxygen consumption (mg/L) and the calculated percentages biodegradation (BOD/ThOD) of sodium acetate and the test substance in the Closed Bottle test. Biodegradation of the test substance is calculated both without nitrification (BOD/ThOD_NH3) and with nitrification (BOD/ThOD_NO3) 

Time (days)	Oxygen consumption (mg/L)		Biodegradation (%)
	Test substance	Acetate	Test substance		Acetate
			ThODNH3	ThODNO3
0	0.00	0.00	0	0	0
7	0.15	3.95	3	3	76
14	1.55	3.90	28	26	75
21	2.85		52	48
28	3.70		67	63

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Remarks:

(>60% after 28 days); these results support mineralization of the test substance (only at a slower rate).

Conclusions:

Under the study conditions, the test substance was determined to be readily biodegradable with 63% biodegradation after 28 days.

Executive summary:

A study was conducted to determine the ready biodegradability of the test substance, TMAC T (96% active), using Closed bottle test, according to the OECD Guideline 301D, in compliance with GLP. Secondary activated sludge was obtained from the domestic wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. The measured dry weight of the inoculum was 3.2 g/L. The activated sludge was preconditioned to reduce the endogenous respiration rates. The preconditioned inoculum was diluted further to a dry weight concentration of 2 mg/L in the BOD bottles. The inoculum concentration in the BOD bottles determined by colony count was 1E+6 CFU/L. The test substance (2 mg/L) was exposed to activated sludge, which was spiked to a mineral nutrient solution, dosed in closed bottles supplemented with 1 g silica gel/bottle as sorbent for detoxification of the test substance, and incubated in the dark at 22.7 to 22.9°C for 28 days. Use was made of 10 bottles containing only inoculum, 10 bottles containing inoculum, silica gel and isopropanol, 10 bottles containing inoculum and silica gel with test substance, 6 bottles containing inoculum and sodium acetate. The concentrations of the test substance, and sodium acetate in the bottles were 2.0 mg/L and 6.7 mg/L, respectively. The concentration isopropanol added to the control bottles with silica gel was 35 µg/L which is comparable to the isopropanol content present in the test bottles. Each of the prepared solutions was dispensed into the respective group of biochemical oxygen demand (BOD) bottles so that all bottles were completely filled without air bubbles. The zero-time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at Day 7, 14, 21, and 28. Endogenous respiration, theoretical oxygen demand (ThOD), biochemical oxygen demand (BOD) and biodegradation were calculated. The degradation of the test substance was assessed by the measurement of oxygen consumption. The ThODNH3 and ThODNO3 of the test substance used to calculate the biodegradation percentages is 2.86 and 3.06 g oxygen/g active ingredient, respectively. According to the results of this study, the test substance did not cause a reduction in the endogenous respiration at Day 7. The test substance in the presence of silica gel is therefore considered to be non-inhibitory to the inoculum in the test. The test substance was biodegraded by 67% (based on ThODNH3), at Day 28. Assuming a complete nitrification of the organic nitrogen present in the test substance and using a correction for the oxygen consumption by the nitrification, the test substance was biodegraded by 63% at day 28 (based on ThODNO3). The validity of the test is demonstrated by an endogenous respiration of 1.10 mg/L at Day 28. Furthermore, the differences of the replicate values at Day 28 were less than 20%. The biodegradation percentage of the reference compound, sodium acetate, at Day 14 was 75%. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period Under the study conditions, the test substance was determined to be readily biodegradable with >60% biodegradation after 28 days (Geerts, 2020).

Reference 4

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

From October 4, 2005 to November 02, 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Information on the category justification can be found in the Quaternary ammonium salts (QAS) category and section 13.2 of IUCLID.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

yes

Remarks:

The test was modified to permit prolonged measurements (van Ginkel and Stroo 1992).

Qualifier:

according to guideline

Guideline:

EU Method C.6 (Degradation: Chemical Oxygen Demand)

Version / remarks:

Degradation-biotic degradation: Closed Bottle test

Deviations:

yes

Remarks:

The test was modified to permit prolonged measurements (van Ginkel and Stroo 1992).

Qualifier:

according to guideline

Guideline:

ISO 10707 Water quality - Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds - Method by analysis of biochemical oxygen demand (closed bottle test)

Deviations:

yes

Remarks:

The test was modified to permit prolonged measurements (van Ginkel and Stroo 1992).

Principles of method if other than guideline:

The test was modified to permit prolonged measurements (van Ginkel and Stroo 1992).

GLP compliance:

yes

Specific details on test material used for the study:

CAS number: 112-03-8
Purity: 99.5%
Appearance: White powder

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, adapted

Details on inoculum:

Secondary activated sludge was obtained from the WWTP Nieuwgraaf in Duiven, The Netherlands. The WWTP Nieuwgraaf is an activated sludge plant treating predominantly domestic waste water. A minor deviation of the test procedures described in the guidelines was introduced: instead of an effluent/extract/mixture, activated sludge was used as an inoculum. The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 400 mg Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted to a concentration of 2 mg DW/L in the BOD bottles (van Ginkel and Stroo 1992).

Duration of test (contact time):

28 d

Initial conc.:

1 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

The oxygen concentration was measured with a special funnel which enabled testing without sacrificing bottles. This funnel exactly fitted into the BOD bottle. Subsequently, the oxygen electrode was inserted into the BOD bottle to measure the oxygen concentration. The medium dissipated by the electrode was collected in the funnel. After withdrawal of the oxygen electrode the medium collected flew back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo, 1992). The oxygen concentrations were measured in quadruplicate bottles instead of the prescribed duplicate bottle to improve accuracy. Use was therefore made of 4 bottles containing only inoculum, 4 bottles containing test substance and inoculum, and 4 bottles containing sodium acetate and inoculum. The concentrations of the test substance and sodium acetate in the bottles were 1.0 and 6.7 mg/L, respectively. The inoculum was diluted to 2 mg DW/L in the closed bottles. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles.

Reference substance:

acetic acid, sodium salt

Remarks:

concentration in the bottles: 6.7 mg/L

Preliminary study:

Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test substance in the Closed Bottle test was not determined because possible toxicity of the test substance to microorganisms degrading acetate is not relevant. A slight inhibition of the endogenous respiration of the inoculum by the test substance was detected at day 7. Therefore, limited inhibition of the biodegradation due to the "high" initial concentration of the test substance is expected. This toxicity was the reason for testing at an initial test substance concentration of 1.0 mg/L.

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

ratio BOD/ThoD

Value:

77

Sampling time:

28 d

Remarks on result:

other: readily biodegradable

Details on results:

The calculated theoretical oxygen demand of the test substance was 2.9 mg/mg. This theoretical oxygen demand is calculated by assuming formation of ammonium chloride.
The pH of the media was 7.0 at the start of the test. The pH of the medium at Day 28 was 6.8. Temperatures ranged from 19 to 21°C.

Results with reference substance:

The ThOD of sodium acetate was 0.8 mg/mg.
The biodegradation percentage at Day 14 was 66%.

Validity of the test:

The validity of the test is demonstrated by an endogenous respiration of 1.1 mg/L at day 28. Furthermore, the differences of the replicate values at day 28 were less than 20%. The biodegradation percentage of the reference substance, sodium acetate, at day 14 was 66. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Under the study conditions, the biodegradation of the source substance was determined to be 77% and the target substance was therefore considered readily biodegradable (activated sludge, domestic).

Executive summary:

A study was conducted to determine the biodegradation in water of the source substance, TMAC C18 (99.5% active) according to OECD guideline 301D, EU Method C.6 and ISO 10707 (Closed Bottle test), in compliance with GLP. The test was performed with activated sludge, domestic in 0.30L BOD (biological oxygen demand) bottles with glass stoppers. There were 10 bottles containing only river water, 6 bottles containing river water and sodium acetate, 10 bottles containing river water with the source substance. The concentrations of the source substance, and sodium acetate in the bottles were 1.0, and 6.7 mg/L, respectively. (A slight inhibition of the endogenous respiration of the inoculum by the source substance was detected at day 7. Therefore, limited inhibition of the biodegradation due to the "high" initial concentration of the test compound is expected. This toxicity was the reason for testing at an initial test compound concentration of 1.0 mg/L). The source substance was biodegraded by 77% and 73% by the end of 28 days using and ThODNH3 and ThODNO3 equations respectively. The test was valid, as shown by an endogenous respiration of 1.1 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 66% of its theoretical oxygen demand after 14 day. Oxygen concentrations remained >0.5 mg/ L in all bottles during the test period. Under the study conditions, the source substance can be considered readily biodegradable (van Ginkel, 2005). Based on the results from this longer chain alcohol free quaternary ammonium substance, which would represent a worst case – as longer chains tend to biodegrade more slowly than shorter chains, the target substance, which is mix of C16 and C18 alkyl chains, can be expected to be degrading faster than the source substance.

Description of key information

Overall, considering all the above information together, the registered substance is considered to be readily biodegradable undergoing complete mineralization.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

Study 1:A preliminary non-GLP study was conducted to determine the best test conditions for conducting the closed bottle ready biodegradation study with the target substance, TMAC T (96% active), according to the OECD Guideline 301D. Due to the well-known toxicity of the quaternary substances, the target substance was evaluated using detoxification methods through the addition of the sorbents silica gel and humic acid at two different concentrations. Activated sludge or river water was used as inoculum in the Closed Bottle test. In addition, a sorbent free test group without any deviations from the guideline was included as a ‘negative control’, to demonstrate the toxicity of the target substance and to demonstrate the positive detoxifying effects of the sorbents. Ammonium chloride was omitted from the medium to prevent nitrification for all groups except the sorbent free group. The inoculum concentration in the bottles determined by colony count was 7E+5 CFU/L and 6E+5 CFU/L for the river water and activated sludge inoculum, respectively. The tests were performed in triplicates using 0.3 L BOD bottles with glass stoppers. In the tests ‘without sorbent’ use was made of 3 bottles with the target substance (at 2 mg/L) and the respective inoculum and 3 control bottles only containing the respective inoculum and 36 μg/L isopropanol (to correct for the small amount of isopropanol still present in the target substance). In the ‘sorbent modified’ tests use was made of 3 bottles containing the target substance (at 2 mg/L), the respective inoculum and silica gel or humic acid, and 3 control bottles containing only respective inoculum, 36 μg/L isopropanol, and silica gel or humic acid. Silicagel and humic acid concentrations in the bottles (test and control) were 1 and 2 g /bottle and 1 and 2 mg acid/L, respectively. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The bottles were closed and incubated in the dark at temperatures ranging from 22 to 24°C. The biodegradation was measured by following the course of the oxygen decrease in the bottles using a special funnel and an oxygen electrode. The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter (WTW). The theoretical oxygen demand (ThOD) of target substance was calculated from its molecular formula and molecular weight. The BOD (mg/mg) of the target substance was calculated by dividing the oxygen consumption by the concentration of the target substance in the closed bottle. The ThODNH3 and ThODNO3 of the active ingredient (active with average chain length) used to calculate the biodegradation percentages was 2.86 g/g and 3.06 g/g, respectively. The biodegradation percentages at Day 28 using activated sludge as inoculum were slightly higher compared to results achieved with river water. Using the conservative ThODNO3 to calculate the biodegradation of target substance still >60% biodegradation was achieved within 28 days using activated sludge as inoculum and 1 g silica gel / bottle for detoxification. The validity of the test is demonstrated by oxygen concentrations >0.5 mg/L in all bottles during the test period. The pH of the media was 7.4 and 7.2±0.1 (activated sludge) and 8.2 and 8.0±0.1 (river water) at the start and end of Day 42 of the test respectively. Temperatures ranged from 22 to 24°C. The inhibition of biodegradation by the target substances is usually detected prior to the onset of the biodegradation through suppression of the endogenous oxygen consumption and this was clearly detected until day 7-14 in the sorbent free ready biodegradation tests. The humic acid sorbent still showed an inhibition of the endogenous respiration (negative biodegradation percentages) at Day 7. Detoxification was most successful by the silica gel sorbents and no inhibition of the biodegradation due to the “high” initial target substance concentration is expected in the presence of silica gel (1 and 2 g/bottle). Under the study conditions, the target substance was determined to be readily biodegradable and the use activated sludge as inoculum and 1 g silica gel /bottle for detoxification of the target substance was considered further for the main study (Geerts, 2020).

The main study was conducted to determine the ready biodegradability of the target substance, TMAC T (96% active), using Closed bottle test, according to the OECD Guideline 301D, in compliance with GLP. Secondary activated sludge was obtained from the domestic wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. The measured dry weight of the inoculum was 3.2 g/L. The activated sludge was preconditioned to reduce the endogenous respiration rates. The preconditioned inoculum was diluted further to a dry weight concentration of 2 mg/L in the BOD bottles. The inoculum concentration in the BOD bottles determined by colony count was 1E+6 CFU/L. The target substance (2 mg/L) was exposed to activated sludge, which was spiked to a mineral nutrient solution, dosed in closed bottles supplemented with 1 g silica gel/bottle as sorbent for detoxification of the target substance, and incubated in the dark at 22.7 to 22.9°C for 28 days. Use was made of 10 bottles containing only inoculum, 10 bottles containing inoculum, silica gel and isopropanol, 10 bottles containing inoculum and silica gel with target substance, 6 bottles containing inoculum and sodium acetate. The concentrations of the target substance, and sodium acetate in the bottles were 2.0 mg/L and 6.7 mg/L, respectively. The concentration isopropanol added to the control bottles with silica gel was 35 µg/L which is comparable to the isopropanol content present in the test bottles. Each of the prepared solutions was dispensed into the respective group of biochemical oxygen demand (BOD) bottles so that all bottles were completely filled without air bubbles. The zero-time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at Day 7, 14, 21, and 28. Endogenous respiration, theoretical oxygen demand (ThOD), biochemical oxygen demand (BOD) and biodegradation were calculated. The degradation of the target substance was assessed by the measurement of oxygen consumption. The ThODNH3 and ThODNO3 of the target substance used to calculate the biodegradation percentages is 2.86 and 3.06 g oxygen/g active ingredient, respectively. According to the results of this study, the target substance did not cause a reduction in the endogenous respiration at Day 7. The target substance in the presence of silica gel is therefore considered to be non-inhibitory to the inoculum in the test. The target substance was biodegraded by 67% (based on ThODNH3), at Day 28. Assuming a complete nitrification of the organic nitrogen present in the target substance and using a correction for the oxygen consumption by the nitrification, the target substance was biodegraded by 63% at day 28 (based on ThODNO3). The validity of the test is demonstrated by an endogenous respiration of 1.10 mg/L at Day 28. Furthermore, the differences of the replicate values at Day 28 were less than 20%. The biodegradation percentage of the reference compound, sodium acetate, at Day 14 was 75%. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period Under the study conditions, the target substance was determined to be readily biodegradable with >60% biodegradation after 28 days (Geerts, 2020).

Study 2:A study was conducted to determine the ready biodegradability of the target substance, TMAC T (49% active in isopropanol and water), in water according to OECD Guideline 301D (closed bottle test), in compliance with GLP. The target substance at 2 mg/L was incubated with inoculums from river water and O2 consumption was followed over a period of 28 days. The test was performed using 10 bottles containing only river water (inoculum and medium), 10 bottles containing river water and silica gel (2 g/bottle), 10 bottles containing river water, silica gel and target substance, and 6 bottles containing sodium acetate and river water. The concentrations of the target substance and sodium acetate in the bottles were 2.0 and 6. 7 mg/L, respectively. Each of the prepared solutions was dispensed into the respective group of 0.3 L BOD bottles so that all bottles were completely filled without air bubbles. The zero-time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at Day 7, 14, 21, and 28. The test was found to be valid as shown by an endogenous respiration of 1.0 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 76% of its theoretical oxygen demand after 14 day. Finally, the most important criterion was met with the oxygen concentrations being > 0.5 mg/L in all bottles during the test period. Biodegradability was determined to be 71% and 69% by the end of 28 days using and ThODNH3 and ThODNO3 equations respectively. Therefore, the substance can be considered readily biodegradable in water. Furthermore, the target substance did not cause a reduction in the endogenous respiration in presence of silica gel, hence was considered to be non-inhibitory to the inoculum. Under the conditions of the study, the target substance is considered to be readily biodegradable (van Ginkel CG, 2010).

Study 3:A study was conducted to determine the biodegradation in water of the source substance, TMAC C18 (99.5% active) according to OECD guideline 301D, EU Method C.6 and ISO 10707 (Closed Bottle test), in compliance with GLP. The test was performed with activated sludge, domestic in 0.30L BOD (biological oxygen demand) bottles with glass stoppers. There were 10 bottles containing only river water, 6 bottles containing river water and sodium acetate, 10 bottles containing river water with the source substance. The concentrations of the source substance, and sodium acetate in the bottles were 1.0, and 6.7 mg/L, respectively. (A slight inhibition of the endogenous respiration of the inoculum by the source substance was detected at day 7. Therefore, limited inhibition of the biodegradation due to the "high" initial concentration of the test compound is expected. This toxicity was the reason for testing at an initial test compound concentration of 1.0 mg/L). The source substance was biodegraded by 77% and 73% by the end of 28 days using and ThODNH3 and ThODNO3 equations respectively. The test was valid, as shown by an endogenous respiration of 1.1 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 66% of its theoretical oxygen demand after 14 day. Oxygen concentrations remained >0.5 mg/ L in all bottles during the test period. Under the study conditions, the source substance can be considered readily biodegradable (van Ginkel, 2005). Based on the results from this longer chain alcohol free quaternary ammonium substance, which would represent a worst case – as longer chains tend to biodegrade more slowly than shorter chains, the target substance, which is mix of C16 and C18 alkyl chains, can be expected to be degrading faster than the source substance.

The use of silica gel in the key study on biodegradation is supported by the findings from van Ginkel 2008, which showed that silica gel was the best adsorbent as compared to lignosulphonic acid and humic acid (seeFigure 1 in the CSR):

 

In addition, recent publications from Timmeret al.,2019 and Nabeokaet al.,2020 indicate that use of appropriate concentrations of moderate adsorbent carriers like silica gel has the ability to reduce the microbial toxicity of quaternary ammonium substances (by lowering their concentrations) and hence increasing their biodegradation. However, the use of silica gel was found to have no effect on highly persistent substances with specific chemical structures, e.g., branched alkyl chain containing substances as in benzethonium chloride (Nabeoka et al., 2020). This is a critical observation as it demonstrates that use of silica gel in the studies with the linear alkyl chain containing quaternary substances like the target substance does not overestimate the biodegradation.

Further, the results obtained with the target substance are in agreement with what is reported in the literature for other quaternary ammonium substances, as summarized below inTable 4.4.

Table 4.4. Compilation of ready biodegradability test results obtained with quaternary ammonium salts (adapted van Ginkel, 2007)

Substance	Test	Results at Day 28 (%)
Hexadecyltrimethylammonium Chloride (TMAC C16)	Headspace Carbon Dioxide	75*
Octadecyltrimethylammonium Chloride (TMAC C18)	Sturm test	>70
Cocotrimethylammonium (TMAC C)	Closed bottle	>60
Octylbenzyldimethylammonium chloride (C18 ADBAC)	MITI	>80
Tetradecylbenzyldimethylammonium Chloride (C14 ADBAC)	MITI	>80
Decylbenzyldimethylammonium Chloride (C10 ADBAC)	Closed bottle	>60

*Mean from 10 laboratories; also cited in OECD TG 310 (adopted on 23 March 2006)

Biodegradation pathways of quaternary substances

In addition, several literature data are available to clarify the metabolic basis of degradation by micro-organisms. Bacteria identified as Pseudomonas sp capable of degrading alkyltrimethylammonium salts were isolated from activated sludge (van Ginkelet al., 1992; Takenakaet al., 2007). Alkyltrimethylammonium salts with octadecyl, hexadecyl, tetradecyl, dodecyl, decyl, octyl, hexyl and coco alkyl chains supported growth of the isolates, showing the broad substrate specificity with respect to the alkyl chain length. Alkanals, and fatty acids can also serve as a carbon and energy source (van Ginkelet al., 1992; Takenakaet al., 2007). In simultaneous adaptation studies,1H nuclear magnetic resonance spectrometry (1H-NMR) and GC-MS showed that acetate, alkanals and alkanoates are the main intermediates of alkyltrimethylammmonium salt degradation, indicating that the long alkyl chain is utilized for microbial growth (van Ginkelet al., 1992; Nishiyama and Nishihara, 2002; Takenakaet al., 2007). Trimethylamine is stoichiometrically produced by pure cultures of microorganisms growing with the alkyl chain of alkyltrimethylammonium chloride as the sole source of carbon. The cleavage of the C-alkyl-N bond of alkyltrimethylammonium salts resulting in the formation of trimethylamine is initiated by a mono-oxygenase (van Ginkelet al., 1992). Additional evidence of the cleavage of the C-alkyl-N bond as the initial degradation step of alkyltrimethylammonium salts was presented by Nishiyamaet al. (1995) and Takenakaet al. (2007).

Dehydrogenase activity present in cell-free extract of hexadecyltrimethylammonium chloride-grown cells catalysed the oxidation of alkanal to fatty acids. The route of the fatty acid degradation is by β-oxidation. Trimethylamine, a naturally occurring compound is readily biodegradable (Pitter and Chudoba 1990). Complete degradation of trimethylamine is demonstrated through the assessment of the biodegradation pathway. Trimethylamine is degraded by methylotrophic bacteria through successive cleavage of the methyl groups (Large, 1971; Meiberg and Harder, 1978). Consortia of microorganisms degrading the alkyl chain of alkyltrimethylammonium salts and trimethylamine are therefore capable of complete (ultimate) degradation of alkyltrimethylammonium salts. Complete degradation of alkyltrimethylammonium salts using a mixed culture has been demonstrated by Nishiyamaet al. (1995). More recently, Nishiyama and Nishihara (2002) have isolated aPseudomonas spcapable of degrading both the alkyl chain and trimethylamine.  Both the pure and mixed culture studies showed that the degradation of the alkyl chain of alkyltrimethylammonium salts results in the formation of water, carbon dioxide and ammonium (seeFigure 2in the CSR).

Further, according to the evidence presently available on the biodegradation rate, microorganisms readily oxidize the hydrophobic alkyl chains of the cationic surfactants, which is followed by a slower oxidation of the hydrophilic moiety (the corresponding amines) (van Ginkel, 2004). The above biodegradation process for the two moieties plays a key role in the differences in the results between the different cationic surfactants. However, based on the available experimental data and literature evidence, the alkyl chains and the trimethylamine of the target substance is readily biodegradable.

Overall, considering all the above information together, the target substance is considered to be readily biodegradable undergoing complete mineralization